Bleaching herbicide norflurazon inhibits phytoene desaturase by competition with the cofactors.

Cofactor requirement was determined for the heterologous expressed phytoene desaturases from the cyanobacterium Synechococcus and the higher plant Gentiana lutea. The cyanobacterial enzyme is dependent on either NAD(P) or plastoquinone, whereas only quinones such as plastoquinone can function as a cofactor for the phytoene desaturase from G. lutea. Enzyme kinetic studies were carried out to determine a possible competition between the cofactors and the bleaching herbicide norflurazon. For the Synechococcus enzyme, competition between norflurazon and NADP, as well as plastoquinone, could be demonstrated. The K(m) values for these cofactors were 6.6 mM and 0.23 microM, respectively. Inhibition of the phytoene desaturase from G. lutea by norflurazon was also competitive with respect to plastoquinone. The K(m) values of both enzymes for plastoquinone were very close.     

Effect of a tomato-based drink on markers of inflammation, immunomodulation, and oxidative stress

Regular consumption of tomato and its products is being consistently associated with lower risk of several types of cancer and, to a lesser extent, coronary heart disease. Among the many tomato components credited with healthful properties, carotenoids and particularly lycopene are being actively investigated. Given the recognized role of immune/inflammatory processes in atherogenesis, the effects of a tomato-based drink (Lyc-o-Mato), which was previously shown to afford DNA protection from oxidative stress, on the modulation of immune and inflammatory markers (by enzyme immunoessay), on basal lymphocyte DNA damage (by comet assay), and on F2-isoprostane excretion (by LC-MS/MS), were investigated in 26 healthy young volunteers. In a placebo-controlled, double-blind, crossover study, Lyc-o-Mato (5.7 mg of lycopene, 3.7 mg of phytoene, 2.7 mg of phytofluene, 1 mg of beta-carotene, and 1.8 mg of alpha-tocopherol) or a placebo drink (same taste and flavor, but devoid of active compounds) were given for 26 days, separated by a wash-out period. During the study subjects maintained their habitual, hence unrestricted, diet. TNF-alpha production by whole blood was 34.4% lower after 26 days of drink consumption, whereas the other parameters were not significantly modified by the treatment. In turn, modest effects of the regular intake of a tomato drink, providing small amounts of carotenoids, were found on the production of inflammatory mediators, such as TNF-alpha, in young healthy volunteers. Future intervention trials in subjects with low carotenoid status and/or compromised immune system will resolve the issue of whether carotenoids modulate immune parameters in humans.     

In vitro and in situ inhibition of carotenoid biosynthesis in Capsicum annuum by bleaching herbicides

Pepper leaves treated with the herbicide J852 show an accumulation of phytoene and zeta-carotene, whereas treatment with norflurazon led to an accumulation of only phytoene. The effects of these herbicides were examined in vitro after the expression of carotenoid desaturases in Escherichia coli. Whereas norflurazon is a potent inhibitor of phytoene desaturase (PDS) (I(50) = 0.12 microM) but not of zeta-carotene desaturase (ZDS) (I(50) = 144 microM), J852 inhibits both PDS (I(50) = 23 microM) and ZDS (I(50) = 49 microM). The influence of PDS/ZDS inhibition on gene expression was examined by comparative RT-PCR. None of the examined genes, namely, encoding phytoene synthase, PDS, ZDS, or the terminal oxidase associated with phytoene desaturation, were induced upon herbicide treatment in pepper leaves or seedlings. This was unexpected because inhibition of carotene desaturation led to an up-regulation of the carotenoid biosynthetic capacity (higher amounts of accumulating precursors plus remaining colored carotenoids are present in treated tissues versus control).     

Processing effects on lycopene content and antioxidant activity of tomatoes

Consumption of tomato products has been associated with decreased risk of some cancer types, and the tomato antioxidant, lycopene, is thought to play an important role in the observed health effects. In this study, four carotenoids, trans-lycopene, phytofluene, phytoene, and zeta-carotene, were quantified in tomato products. Samples of raw tomatoes, tomato juice after hot break scalder, and final paste were obtained from two different processing plants over two years. Comparison of carotenoid levels throughout processing indicated that lycopene losses during processing of tomatoes into final paste (25-30 degrees Brix) ranged from 9 to 28%. The initial Brix level of the raw tomatoes appeared to influence the amount of lycopene loss that occurred, possibly due to the differences in processing time required to achieve the final desired Brix level of the paste. In general, no consistent changes in the other carotenoids were observed as a function of processing. The antioxidant activity of fresh tomatoes, tomato paste, and three fractions obtained from these products (i.e., aqueous, methanol, and hexane fractions) was also determined. In both a free radical quenching assay and a singlet oxygen quenching assay, significant antioxidant activity was found in both the hexane fraction (containing lycopene) and the methanol fraction, which contained the phenolic antioxidants caffeic and chlorogenic acid. The results suggest that in addition to lycopene, polyphenols in tomatoes may also be important in conferring protective antioxidative effects.     

Optimization of lycopene extraction from tomato cell suspension culture by response surface methodology

Radioisotope-labeled lycopene is an important tool for biomedical research but currently is not commercially available. A tomato cell suspension culture system for the production of radioisotope-labeled lycopene was previously developed in our laboratory. In the current study, the goal was to optimize the lycopene extraction efficiency from tomato cell cultures for preparatory high-performance liquid chromatography (HPLC) separation. We employed response surface methodology (RSM), which combines fractional factorial design and a second-degree polynomial model. Tomato cells were homogenized with ethanol, saponified by KOH, and extracted with hexane, and the lycopene content was analyzed by HPLC-PDA. We varied five factors at five levels: ethanol volume (1.33-4 mL/g); homogenization period (0-40 s/g); saturated KOH solution volume (0-0.67 mL/g); hexane volume (1.67-3 mL/g); and vortex period (5-25 s/g). Ridge analysis by SAS suggested that the optimal extraction procedure to extract 1 g of tomato cells was at 1.56 mL of ethanol, 28 s homogenization, 0.29 mL of KOH, 2.49 mL of hexane, and 17.5 s vortex. These optimal conditions predicted by RSM were confirmed to enhance lycopene yield from standardized tomato cell cultures by more than 3-fold.     

Growth and anthraquinone production of Morinda elliptica cell suspension cultures in a stirred-tank bioreactor

The effects of medium strategy, number of impellers, aeration mode, and mode of operation on Morinda elliptica cell suspension cultures in a stirred-tank bioreactor are described. A lower number of impellers and continuous aeration contributed toward high cell growth rate, whereas a higher number of impellers reduced cell growth rate, although not anthraquinone yield. The semicontinuous mode could indirectly imitate the larger scale version of production medium strategy and improved anthraquinone production even with 0. 012% (v/v) antifoam addition. Production medium promoted both growth (maximum dry cell weight of 24.6 g/L) and anthraquinone formation (maximum content of 19.5 mg/g of dry cell weight), without any necessity for antifoam addition. Cultures in production medium or with higher growth rate and anthraquinone production were less acidic than cultures in growth medium or with lower growth rate and anthraquinone production. Using the best operating variables, growth of M. elliptica cells (24.6 g/L) and anthraquinone yield (0.25 g/L) were 45% and 140%, respectively, lower than those using a shake flask culture after 12 days of cultivation.     

Antiproliferative activity of apples is not due to phenolic-induced hydrogen peroxide formation

Anticancer compound screening of natural products using tumor cell lines has been commonly used to identify anticancer drugs. Two highly significant anticancer drugs, paclitaxel (Taxol) and camptothecin, were discovered using tumor cell lines by the U.S. National Cancer Institute (NCI) screening program of plants. It has been recently reported that the inhibition of cancer cell proliferation by fruit extracts was indirectly caused by phenolic-induced H(2)O(2) production in the cell culture media, suggesting that many previously reported effects of flavonoids and phenolic compounds on cultured cells might be from an artifact of H(2)O(2)-induced oxidative stress. The objective of the present study was to determine if apple extracts induced H(2)O(2) formation in common cell culture media and to investigate if the antiproliferative activity of apple extracts was due to phenolic-induced H(2)O(2) formation. It is reported here that apple extracts did not induce H(2)O(2) formation in WME, DMEM, or DMEM/Ham F12 media with the cell culture conditions tested. These same extracts inhibited proliferation of HepG(2) and Caco-2 cells. Therefore, antiproliferative activity of apple extracts was not due to the phenolic-induced H(2)O(2) production in cell culture media. In addition, H(2)O(2) added to the culture medium at 100 microM did not cause inhibition of cell proliferation in either HepG(2) liver cancer cells or Caco-2 colon cancer cells in vitro.     

Biosynthesis and characterization of 14C-enriched flavonoid fractions from plant cell suspension cultures

A range of radiolabeled anthocyanins, proanthocyanidins, and other flavonoids were accumulated by cell suspension cultures of two plant species, ohelo (Vaccinium pahalae) and grape (a Vitis hybrid, Bailey Alicant A), after providing uniformly labeled [(14)C]sucrose to the medium. Approximately 15% of administered label was recovered in a series of flavonoid-rich fractions varying in composition. Anthocyanins, and monomers to oligomers of proanthocyanidins, were labeled effectively and characterized from both species. Most of the proanthocyanidin oligomers were based on the flavan-3-ols (+)-catechin and (-)-epicatechin. Cyanidin and peonidin glycosides were the dominant forms of anthocyanins in both species. Whereas the predominant form of flavonoids identified from ohelo cell cultures was proanthocyanidins, grape cell cultures produced mostly anthocyanins. The labeled phytochemicals were produced for use in subsequent in vivo animal feeding studies to gauge their bioavailability and accumulation in target organs.     

Effects of elicitors on the production of resveratrol and viniferins in cell cultures of Vitis vinifera L. cv Italia

Methyl jasmonate, jasmonic acid and chitosan were tested as elicitors on cell suspension cultures obtained from Vitis vinifera cv Italia to investigate their effect on stilbene production. Stilbene accumulation in the callus, grown under nonelicited conditions, was also investigated. Calli and cell suspensions were obtained in a B5 culture medium supplemented with 0.2 mg L(-1) NAA and 1 mg L(-1) KIN. Stilbene determination was achieved by HPLC/DAD/MS. Whereas callus biosynthesized only piceid, cell suspensions elicited with jasmonates produced several stilbenes, mainly viniferins. In suspended cells, methyl jasmonate and jasmonic acid were the most effective in stimulating stilbene biosynthesis, whereas chitosan was less effective; in fact, the amount of stilbenes obtained with this elicitor was not significantly different from that obtained for the control cells. The maximum production of total stilbenes was at day 20 of culture with 0.970 and 1.023 mg g(-1) DW for MeJA and JA, respectively.     

Chemical and biological characterization of cinnamic acid derivatives from cell cultures of lavender (Lavandula officinalis) induced by stress and jasmonic acid

Cell cultures of lavender (Lavandula officinalis) were analyzed for the metabolite profile under normal growth conditions and under stress as well as after jasmonic acid treatment. The main compound synthesized was rosmarinic acid, which was also secreted into the culture medium. Different solvent extraction methods at different pH values altered the profile slightly. Anoxic stress induced the synthesis of a cinnamic acid derivative, which was identified as caffeic acid by gas chromatography-mass spectrometry. Caffeic acid was also induced after treatment of the cell cultures with jasmonic acid. Although the antioxidative activity of both compounds, rosmarinic acid and caffeic acid, was confirmed in an assay using 2,2-diphenyl-1-picrylhydrazyl (DPPH), it was demonstrated that both substances have a low cytotoxic potential in vitro using acute myeloid leukemia (HL-60) cells. The potential of the system for finding new bioactive compounds is discussed.     

植酸对霍山石斛类原球茎悬浮培养细胞生长和多糖合成的影响

研究了植酸对霍山石斛类原球茎悬浮培养细胞生长、多糖合成和培养基中主要营养成分消耗的影响,并分析了细胞中可溶性还原糖、可溶性蛋白质、丙二醛含量以及过氧化物酶和多酚氧化酶的活性。结果表明,植酸能抑制过氧化物酶和多酚氧化酶的活性,提高细胞活力,从而促进细胞生长和多糖合成,以2.5g/L浓度的植酸效果最好,培养36d时,类原球茎干重为29.4g/L,多糖产量为2.06g/L。动力学分析表明,添加2.5g/L的植酸还有利于碳、氮、磷等营养物质的吸收。     

Metabonomic strategy for the investigation of the mode of action of the phytotoxin (5S,8R,13S,16R)-(-)-pyrenophorol using 1H nuclear magnetic resonance fingerprinting

The biochemical mode of action of (5S,8R,13S,16R)-(-)-pyrenophorol isolated from a Drechslera avenae pathotype was investigated by using metabolic fingerprinting. (1)H NMR spectra of crude leaf extracts from untreated Avena sterilis seedlings and A. sterilis seedlings treated with pyrenophorol were compared with those obtained from treatments with the herbicides diuron, glyphosate, mesotrione, norflurazon, oxadiazon, and paraquat. Multivariate analysis was carried out to group treatments according to the mode of action of the phytotoxic substances applied. Analysis results revealed that none of the herbicide treatments fitted the pyrenophorol model and indicate that the effect of the phytotoxin on A. sterilis differs than those caused by glyphosate, mesotrione, norflurazon, oxadiazon, paraquat, and diuron, which inhibit 5-enolpyruvylshikimate-3-phosphate synthase, 4-hydroxyphenyl-pyruvate-dioxygenase, phytoene desaturase, protoporphyrinogen oxidase, photosystem I, and photosystem II, respectively. The method applied, combined with appropriate data preprocessing and analysis, was found to be rapid for the screening of phytotoxic substances for metabolic effects.     

Eelgrass slabs, a soilless culture substrate that inhibits adhesion of fungi and oomycetes and enhances antioxidant activity in tomato

Composed of a marine plant, Zostera sp., eelgrass slabs are a novel organic substrate for soilless cultures used in tomato production. The benefit of using eelgrass slabs for growing tomatoes was assessed by comparing it with coconut fiber slabs in regard to contamination by Pythium spp. and to the antioxidant properties of tomato fruits. First, tomato root contamination by Pythium spp. was studied by direct plate counting, and a molecular comparison of fungal and oomycete communities was conducted using PCR-DHPLC. Second, the antioxidant properties of tomato fruits were analyzed by measuring total phenol and carotenoid contents and by evaluating radical scavenging activity. Compared to plants grown on coconut fiber slabs, those on eelgrass slabs presented a lower rate of Pythium spp. root contamination. Moreover, culture on eelgrass slabs produced fruits with better radical scavenging activity and higher total phenol content compared to controls. Carotenoid content was not affected by the type of substrate. This study highlights the value of detrital leaves of Zostera sp. as a substrate for soilless culture that reduces root contamination and also promotes the production of tomato fruits with better nutritional value.     

Isolation of radiolabeled isoflavones from kudzu (Pueraria lobata) root cultures

Isoflavones have potential for preventing and treating several chronic health conditions, such as osteoporosis, cardiovascular disease, and cancer. In this study, radiolabeled isoflavones were recovered from kudzu (Pueraria lobata) root cultures after incubation with uniformly labeled (14)C-sucrose in the culture medium for 21 days. Approximately 19% of administered label was recovered in the isoflavone-rich dried extracts of kudzu root cultures (90.2 microCi/g or 3.3 MBq/g extract). HPLC-PDA analysis revealed the predominant isoflavones isolated from kudzu root cultures to be puerarin, daidzin, and malonyl-daidzin. The average concentration of the major isoflavone puerarin in kudzu root cultures was 33.6 mg/g extract, with a specific activity of 63.5 microCi/g (2.3 MBq/g). The isolated isoflavones were sufficiently (14)C-labeled to permit utilization for subsequent in vivo metabolic tracking studies.     

Liquid chromatography-mass spectrometry of cis- and all-trans-lycopene in human serum and prostate tissue after dietary supplementation with tomato sauce

Several epidemiological studies suggest a lower incidence of prostate cancer in men who routinely consume tomato products. Tomatoes are the primary dietary source of lycopene, which is among the most potent antioxidants of the carotenoids. Men with clinical stage T1 or T2 prostate adenocarcinoma were recruited (n = 32) and consumed tomato sauce based pasta dishes for 3 weeks (equivalent to 30 mg of lycopene per day) before radical prostectomy. Prostate tissue from needle biopsy just before intervention and prostectomy after supplementation from a subset of 11 subjects was evaluated for both total lycopene and lycopene geometrical isomer ratios. A gradient HPLC system using a C(18) column with UV-vis absorbance detection was used to measure total lycopene. Because the absorbance detector was insufficiently sensitive, HPLC with a C(30) column and positive ion atmospheric pressure chemical ionization mass spectrometric (LC-MS) detection was developed as a new assay to measure the ratio of lycopene cis/trans isomers in these samples. The limit of detection of the LC-MS method was determined to be 0.93 pmol of lycopene on-column, and a linear response was obtained over 3 orders of magnitude. Total lycopene in serum increased 2.0-fold from 35.6 to 69.9 microg/dL (from 0.664 to 1.30 microM) as a result of dietary supplementation with tomato sauce, whereas total lycopene in prostate tissue increased 3.0-fold from 0.196 to 0.582 ng/mg of tissue (from 0.365 to 1.09 pmol/mg). all-trans-Lycopene and at least 14 cis-isomer peaks were detected in prostate tissue and serum. The mean proportion of all-trans-lycopene in prostate tissue was approximately 12.4% of total lycopene before supplementation but increased to 22.7% after dietary intervention with tomato sauce. In serum there was only a 2.8% but statistically significant increase in the proportion of all-trans-lycopene after intervention. These results indicate that short-term supplementation with tomato sauce containing primarily all-trans-lycopene (83% of total lycopene) results in substantial increases in total lycopene in serum and prostate and a substantial increase in all-trans-lycopene in prostate but relatively less in serum.     

Tomato productivity and the associated composition of the hydroponic or soil solution

The concentration and balance of nutrients in both the hydroponic or soil solution has been evaluated with relevance to tomato productivity. Yields of 200 mt/ha have been produced in greenhouse, hydroponic culture in a 9 to 10 month period. Yields of more than 100 mt/ha of field grown tomatoes have been produced in Florida in a 4 to 5 month period. Within the limitations of either culture, it is possible per unit of time to provide the nutrients required to produce equivalent yields. It is most significant that field grown tomatoes in Florida can be grown with minimal management and at a fraction of the cost of those grown hydroponically. The validity of the Florida fresh market tomato industry is based on the production efficiency of the field culture. There are annually 16,000 ha of tomatoes grown in Florida, perhaps less than 40 ha grown in hydroponic cultures.     

Structure-activity relationships of α-, β(1)-, γ-, and δ-tomatine and tomatidine against human breast (MDA-MB-231), gastric (KATO-III), and prostate (PC3) cancer cells

Partial acid hydrolysis of the tetrasaccharide (lycotetraose) side chain of the tomato glycoalkaloid α-tomatine resulted in the formation of four products with three, two, one, and zero carbohydrate side chains, which were separated by high-performance liquid chromatography (HPLC) and identified by thin-layer chromatography (TLC) and liquid chromatography ion-trap time-of-flight mass spectrometry (LCMS-IT-TOF). The inhibitory activities in terms of IC(50) values (concentration that inhibits 50% of the cells under the test conditions) of the parent compound and the hydrolysates, isolated by preparative HPLC, against normal human liver and lung cells and human breast, gastric, and prostate cancer cells indicate that (a) the removal of sugars significantly reduced the concentration-dependent cell-inhibiting effects of the test compounds, (b) PC3 prostate cancer cells were about 10 times more susceptible to inhibition by α-tomatine than the breast and gastric cancer cells or the normal cells, (c) the activity of α-tomatine against the prostate cancer cells was 200 times greater than that of the aglycone tomatidine, and (d) the activity increased as the number of sugars on the aglycone increased, but this was only statistically significant at p < 0.05 for the normal lung Hel299 cell line. The effect of the alkaloids on tumor necrosis factor α (TNF-α) was measured in RAW264.7 macrophage cells. There was a statistically significant negative correlation between the dosage of γ- and α-tomatine and the level of TNF-α. α-Tomatine was the most effective compound at reducing TNF-α. The dietary significance of the results and future research needs are discussed.     

Monascuspiloin induces apoptosis and autophagic cell death in human prostate cancer cells via the Akt and AMPK signaling pathways

Monascus pigments have been reported to possess anticancer effects in various cancer cells; however, the molecular mechanisms of their anticancer properties remain largely unknown. Monascuspiloin is an analogue of the Monascus pigment monascin, and its anticancer growth activity against human prostate cancer cells was evaluated using in vitro and in vivo models. Monascuspiloin effectively inhibits the growth of both androgen-dependent LNCaP and androgen-independent PC-3 human prostate cancer cells. Monascuspiloin preferentially induces apoptosis in LNCaP cells by attenuating the PI3K/Akt/mTOR pathway. In androgen-independent PC-3 cells, monascuspiloin induces G2/M arrest and autophagic cell death by an AMPK-dependent pathway. Induction of autophagy in PC-3 cells further sensitizes cells to apoptosis induced by monascuspiloin. Monascuspiloin inhibits tumor growth in nude mice bearing PC-3 xenografts through induction of apoptosis and autophagy. This study is the first to demonstrate that monascuspiloin has therapeutic potential for the treatment of both androgen-dependent and -independent human prostate cancers.     

Transformation of proanthocyanidin A2 to its isomers under different physiological pH conditions and common cell culture medium

Proanthocyanidins constitute an important class of polyphenols ubiquitously found in plants. They have been extensively studied for their antioxidant capacity and bioactivity in vitro and in animal models. However, their stability under different pH conditions and in cell culture medium has not been well documented. In the present study, it was observed that proanthocyanidin A2 (PA2) was relatively more stable in acidic condition than in weak alkaline condition. PA2 was also quite unstable in basal-Dulbecco's Modified Eagle medium (b-DMEM medium) at 37 °C. The addition of PA2 to the cell culture medium accelerated its epimerization with a half-life of <15 min, and ethylenediaminetetraacetic acid (EDTA) could not stop the reaction. The results also demonstrated that the major isomers transformed in the weak alkaline condition or cell culture medium at 37 °C were identified as epicatechin-(4β→8; 2β→O→7)-ent-catechin (proanthocyanidin A4) and epicatechin-(4β→6; 2β→O→7)-ent-catechin. The rates of transformation were dependent on the pH or the components of the medium. Therefore, the results obtained for PA2 in the cell culture bioassays, which were usually carried out for 24 h, might not represent the true activity of the original PA2. The stability and transformation of PA2 should be considered when the bioactivity of PA2 is evaluated in a given cell culture system.